Stabilization of RNA during laser capture microdissection by performing experiments under argon atmosphere or using ethanol as a solvent in staining solutions.
The combination of laser capture microdissection (LCM) and gene expression experiments allows cell specific expression profiling, which is decisive in cellular transcriptomic exploration. LCM makes possible the isolation of unique cells or group of cells, but maintaining RNA quality during this process is challenging. Several protocols are available for section preparation, but none of those guarantees the integrity of the RNA during microdissection, and operators are recommended to perform LCM during a limited time. We hypothesized that the cause of RNA degradation during the microdissection time is the presence of water rendering endogenous RNase activity possible. We thus developed two methods that stabilize RNA during microdissection time for up to 90 min. The first one consists of performing LCM under an argon atmosphere, thus preventing tissue rehydration; it is compliant with all existing microdissection protocols. The second one is a new fixation and staining method using ethanol as solvent in all preparatory steps to LCM that enhances fixation and dehydration of samples. We assessed several stains in regard of their effect on tissue morphology and RNA integrity and adjusted an ethanolic staining solution of cresyl violet and eosin Y.
Clément-Ziza
Mathieu
M
INSERM U781, Faculté de Médecine, Hôpital Necker-Enfants Malades, Université René-Descartes, 75015, Paris, France.
Munnich
Arnold
A
Lyonnet
Stanislas
S
Jaubert
Francis
F
Besmond
Claude
C
D001128:Argon / Q000737:chemistry*; D003106:Colon / Q000737:chemistry*; D000431:Ethanol / Q000737:chemistry; D006801:Humans; D018613:Microscopy, Confocal / Q000379:methods*; D012313:RNA / Q000302:isolation & purification*; D020871:RNA Stability
D000431:Ethanol; D012313:RNA; D001128:Argon